Agar medium for the growth of mycobacterium tuberculosis

ABSTRACT

A novel agar medium for the isolation, sub-cultivation, and indirect or direct drug-susceptibility testing of  Mycobacterium tuberculosis  is disclosed. Also disclosed are methods of isolating and growing  Mycobacterium tuberculosis  and methods of drug-resistance screening using the agar medium of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119(e) from U.S. Provisional Application Serial No. 60/190,701, filedMar. 20, 2000, and entitled “New Agar Medium For Mycobacteriumtuberculosis”. The entire disclosure of U.S. Provisional ApplicationSerial No. 60/190,701 is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to a novel agar medium for the isolation,sub-cultivation, and indirect or direct drug-susceptibility testing ofMycobacterium tuberculosis. The invention also relates to methods ofisolating and growing Mycobacterium tuberculosis and to methods ofdrug-resistance screening using the agar medium of the invention.

BACKGROUND OF THE INVENTION

[0003] At first glance, it seems that nothing is new in the cultivationof Mycobacterium tuberculosis. The first attempts of M. tuberculosiscultivation on agar medium go back to the report by Fannie and WalterHesse in 1881. In 1882, Robert Koch used blood serum coagulated on glassslides for M. tuberculosis cultivation. Apparently, he was not too muchconcerned about the biosafety of such a procedure. He later improvedthis method, which was called the “plate technique”, by adding peptone,some salts and glycerol. Also, in 1882, Richard Petri invented the petridish to be used instead of a glass slide. These attempts at cultivationon a transparent type of media were interrupted in 1903 withintroduction of the first egg-based media by Dorset (Dorset, Science.17:374, 1903), followed by a variety of egg-based media recipes(American Trudeau Society, Handbook of Tuberculosis Laboratory Methods,Washington, D.C., 1962; IUAT, Bull Int Union Tuberc Lung Dis. 24:78,1954; Jensen, Abteilung Originale. 125:222-239, 1932; Ogawa et al.,Kekkaku. 24:13-29, 1949; Petragnani, Bollettino dell'Istitutosieroterapico Milanese. 5: 173-185, 1926; Petroff, J. Exp. Med.21:38-42, 1915; Stonebrink, Acta Tuberc. Scand. 35:67-80, 1958).

[0004] Based on systemic studies of the tubercle bacilli requirements,Dubos and Middlebrook in 1947 introduced the first effective agar mediumunder the name “Dubos Oleic Acid Albumin Agar” (Dubos, Amer. Rev.Tuberc. Pulm. Dis., 56:334-345, 1947). It followed by an invention byMiddlebrook and Cohn of the 7H10 agar in 1958 (Middlebrook et al., Amer.J. Publ. Health, 48:844-853, 1958) and 7H11 agar in 1968 (Cohn et al.,Am. Rev. Respir. Dis. 98:295-296,1968). Subsequently, two recipes of aselective agar medium containing four antimicrobials to prevent thegrowth of contaminants (Polymyxin B, Amphotericin B, Carbenicillin,Trimethoprim) were introduced in 1972 (Mitchison et al., J. Med.Microbiol. 5:165-175, 1972) and in 1976 (McClatchy et al., Am. J. Clin.Pathol. 65:412-415, 1976). A powder base for all these media, 7H10 agar,contains agar, combination of seven salts, L-glutamic acid, pyridoxine,biotin, malachite green. It is available commercially from a number ofmanufacturers. Preparation of an agar medium requires addition of theOADC growth supplement, which contains sodium oleate, albumin (bovine,fr. V) dextrose, catalase.

[0005] At the time the 7H10 and 7H11 agar media were developed byMiddlebrook and his colleagues working at the National Jewish Hospitalin Denver, Colo., attempts to use an animal serum as a nutritionalsupplement failed because of inconsistent results with different serumbatches. Therefore, a combination of the only standardized productsavailable at that time, sodium oleate with bovine albumin, were used asthe major component of the OADC supplement. The role of the bovinealbumin is to neutralize the oleic acid, which is toxic to mycobacteria,in case of breakdown of sodium oleate. This composition required a finalpH of 6.8, in spite of the known fact that the optimal pH for M.tuberculosis is 6.2-6.4. Subsequently, the presence of 5-7% of CO₂ inthe incubator is required to protect the growing mycobacteria fromfurther alkalization of the environment due to the release of ammonia bythe growing mycobacteria. Until the time of the present invention, theseculture conditions remained the standard in the art.

[0006] The agar-based medium developed by Middlebrook and colleagues hasa number of advantages over egg-based medium. One of them istransparency, which enables earlier detection of growing colonies. Amongthe major disadvantages of the existing agar media are the cost,difficulties in standardization of the OADC supplement, and the need fora CO₂ incubator. These disadvantages represented the main obstacle inimplementing the agar media outside of the US, especially in developingcountries.

[0007] Timely identification of patients with drug resistance,especially those with multi-drug resistant tuberculosis (MDR-TB), is akey element of any comprehensive program targeting prevention ofepidemics of drug-resistant tuberculosis. Drug susceptibility testing ofthe patients' isolates as a means to address this problem in developingcountries has been considered impractical and unrealistic. A number ofreasons for this, real or fictional, have been given by differentauthors, such as extreme sophistication of the procedure, high cost, andextended turnaround time for laboratory reports.

[0008] Pyrazinamide (PZA) is one of the first-line drugs in the standardtreatment regimen currently used for tuberculosis patients. It is nowmandatory in the United States (man. 12), and it is desirable for anycountry with high prevalence of drug resistance (man. 3) to perform adrug susceptibility test for pretreatment isolates from all newtuberculosis patients, at least with the first-line drugs. A variety oftechniques can be used for susceptibility testing with any of theanti-tuberculosis drugs other than PZA, including the agar proportionmethod in its direct or indirect versions. However, the only approvedtest using PZA in the United States is the radiometric method using thespecial PZA liquid medium with pH 6.0 in the BACTEC-460 system (man.10). This method is quite expensive and may not be affordable for manylaboratories, especially in developing countries. In addition, themethod requires the disposal of a substantial volume of ¹⁴C-radiolabeledculture vials, which is not permitted in many countries. Further, thetest is not fully standardized and may give false-positive andfalse-negative results (man. 4-6). Unlike the agar proportion methodtest for other anti-tuberculosis drugs, the test in a liquid medium doesnot provide any information on the actual proportion of thePZA-resistant bacteria in the patient's bacterial population, and itcannot be used as a direct test (i.e., a pure culture must be isolatedfrom the patient sample prior to testing). Therefore, the totalturnaround time of the method is about four weeks, at best. There hasbeen a suggestion to use the agar proportion method for PZA bysubstituting an ADC supplement for the conventional OADC supplement in apH 5.5 agar medium (man. 1, 2). However, this revised method did notfind its way into the clinical laboratory practice because of very poorgrowth of Mycobacterium tuberculosis isolates a pH 5.5 in theconventional agar medium (man. 11). Therefore, while a number ofalternative methods for susceptibility testing with other drugs arewidely available, the BACTEC method remains the only reliable techniquefor a test with PZA. Moreover, PZA only works at an acidic pH, whichalone can inhibit the growth of M. tuberculosis on conventional medium.

[0009] Therefore, prior to the present invention, there was a need for anew agar medium, which does not require sophisticated or expensivecultivation techniques, which can be produced at a lower cost, and whichcan be used for susceptibility/resistance screening of Mycobacteriumtuberculosis isolates against a wide range of drugs, includingPyrazinamide, with a reasonable turnaround time.

SUMMARY OF THE INVENTION

[0010] The present invention generally relates to a novel agar mediumfor the isolation, growth and screening of Mycobacterium tuberculosis.The agar medium includes: (a) an agar base suitable for growth ofMycobacterium tuberculosis; and, (b) animal serum at a concentration ofbetween about 8% and 12% of the final volume of the agar medium. Themedium is at a pH of between about 6.0 to about 6.25. Preferably, theagar base is selected from the group consisting of Middlebrook and Cohn7H10 and Middlebrook and Cohn 7H11.

[0011] The animal serum can be any suitable animal serum including, butnot limited to, bovine fetal calf serum, calf serum, bovine serum,equine fetal calf serum and equine serum. In one embodiment, the animalserum is at a concentration of between about 8% and 12% of the finalvolume of the agar medium; in another embodiment, the animal serum is ata concentration of between about 9% and 11% of the final volume of theagar medium; in another embodiment, the animal serum is at aconcentration of about 10% of the final volume of the agar medium.

[0012] Preferably, the medium is at a pH of between about 6.0 to about6.25, and another one embodiment, the medium of is at a pH of betweenabout 6.0 to about 6.2; in another embodiment, the medium is at a pH ofbetween about 6.1 and 6.2; and in another embodiment, the medium is at apH of about 6.1.

[0013] The agar medium can include a mixture of antimicrobial agentscomprising polymixin B, carbenicillin, amphotericin B, and trimethoprimlactate. In one aspect, the antimicrobial agents are present at a finalconcentration of: 200 units/ml polymyxin B, 10 μg/ml amphotericin B, 50μg/ml carbenicillin, and 20 μg/ml trimethoprim.

[0014] In one aspect, the medium further comprises an amount of at leastone drug effective for the identification of Mycobacterium tuberculosisorganisms that are susceptible or resistant to the drug. Such a drug caninclude, but is not limited to, isoniazid, streptomycin sulfate,di-hydro-streptomycin, rifampin, pyrazinamide, ethambutol, etionamide,capreomycin sulfate, amikacin, kanamycin sulfate, levofloxacin,p-aminosalicylic acid, D-cycloserine, and/or clofazimine. In one aspect,the drug comprises isoniazid and rifampin, wherein each of the drugs isisolated within a segment of the agar medium. In a preferred embodiment,the isoniazid is present in two segments of the agar medium, and whereineach segment contains a different concentration of the isoniazid. Inanother aspect, the drug is includes: isoniazid, rifampin, pyrazinamideand either of streptomycin sulfate or di-hydro-streptomycin, whereineach of the drugs is isolated within a different segment of the agarmedium. In another aspect, the drug comprises: ethambutol, etionamide,levofloxacin, capreomycin sulfate, and either of amikacin or kanamycinsulfate, wherein each of the drugs is isolated within a differentsegment of the agar medium. In yet another aspect, the drug comprises:p-aminosalicylic acid, D-cycloserine, and clofazimine, wherein each ofthe drugs is isolated within a different segment of the agar medium. Inanother aspect, the drug comprises pyrazinamide.

[0015] Yet another embodiment of the present invention relates to amethod for culturing Mycobacterium tuberculosis, comprising inoculatingan agar medium with a sample containing Mycobacterium tuberculosis. Theagar medium is the agar medium as set forth above. In one aspect, themedium further comprises a mixture of antimicrobial agents comprisingpolymixin B, carbenicillin, amphotericin B, and trimethoprim lactate.Preferably, the antimicrobial agents are incorporated into one half ofthe agar medium in a plate, and the other half of the agar medium in theplate does not contain the antimicrobial agents. In this aspect, theagar medium is inoculated with an undiluted sample obtained from apatient, although the sample can be diluted, for example, by at leastabout 10 fold.

[0016] In one aspect, the sample is a previously isolated sample ofMycobacterium tuberculosis.

[0017] Another embodiment of the present invention relates to a methodfor testing the drug susceptibility of a culture of Mycobacteriumtuberculosis. This method includes the steps of: (a) inoculating an agarmedium as set forth above, with a sample containing Mycobacteriumtuberculosis, wherein the agar medium further comprises an amount of atleast one drug effective for selection against Mycobacteriumtuberculosis organisms that are susceptible to the drug; (b) incubatingthe inoculated agar medium for a time sufficient to detect growth of theMycobacterium tuberculosis in the absence of a growth-inhibiting drug;and, (c) measuring growth of the Mycobacterium tuberculosis on the agarmedium as compared to growth of the Mycobacterium tuberculosis on theagar medium in the absence of the at least one drug. A growth rate ofthe Mycobacterium tuberculosis on the agar medium containing the atleast one drug that is less than a pre-established drug-resistance levelfor the at least one drug, when compared to the growth rate of theMycobacterium tuberculosis on the agar medium in the absence of the atleast one drug, indicates that the Mycobacterium tuberculosis issusceptible to the at least one drug. In one aspect, the pre-establisheddrug-resistance level is 1%, and wherein a growth rate of theMycobacterium tuberculosis on the agar medium containing the at leastone drug that is less than 1% of the growth rate of the Mycobacteriumtuberculosis on the agar medium in the absence of the at least one drug,indicates that the Mycobacterium tuberculosis is susceptible to the atleast one drug. In another aspect, the at least one drug ispyrazinamide, wherein the pre-established drug-resistance level is 10%,and wherein a growth rate of the Mycobacterium tuberculosis on the agarmedium containing the pyrazinamide that is less than 10% of the growthrate of the Mycobacterium tuberculosis on the agar medium in the absenceof the pyrazinamide, indicates that the Mycobacterium tuberculosis issusceptible to pyrazinamide.

[0018] Preferably, the step of incubating is for a time of at leastabout 3 weeks. In one aspect, the step of incubating is for a time offrom between about 3 weeks and about 6 weeks. Preferably, the step ofincubating is performed in the absence of supplemental CO₂.

[0019] The drug to be added to the agar medium can include, but is notlimited to, isoniazid, streptomycin sulfate, di-hydro-streptomycin,rifampin, pyrazinamide, ethambutol, etionamide, capreomycin sulfate,amikacin, kanamycin sulfate, levofloxacin, p-aminosalicylic acid,D-cycloserine, and/or clofazimine. In one aspect, the medium comprisesthe drugs isoniazid and rifampin, and each of the drugs is isolatedwithin a different segment of the agar medium. In this aspect, theisoniazid is preferably present in two different segments of the agarmedium, and wherein each segment contains a different concentration ofthe isoniazid. In this aspect, the agar medium can be directlyinoculated with a sample collected, from a patient. In one embodiment,the sample is undiluted. In another embodiment, the agar medium isinoculated with a sample diluted by at least 10 fold.

[0020] In another aspect of this method, the agar medium comprises thedrugs: isoniazid, rifampin, pyrazinamide and either of streptomycinsulfate or di-hydro-streptomycin, and each of the drugs is isolatedwithin a different segment of the agar medium. In this aspect, the agarmedium is preferably inoculated with a previously isolated culture ofMycobacterium tuberculosis from a sample obtained from a patient.

[0021] In another aspect of this method, the medium comprises the drugs:ethambutol, etionamide, levofloxacin, capreomycin sulfate, and either ofamikacin or kanamycin sulfate, and each of the drugs is isolated withina different segment of the agar medium. In this aspect, the agar mediumis preferably inoculated with a previously isolated culture ofMycobacterium tuberculosis from a sample obtained from a patient.

[0022] In another aspect of this method, the medium comprises the drugs:p-aminosalicylic acid, D-cycloserine, and clofazimine, and wherein eachof the drugs is isolated within a different segment of the agar medium.In this aspect, the agar medium is preferably inoculated with apreviously isolated culture of Mycobacterium tuberculosis from a sampleobtained from a patient.

[0023] In yet another aspect of this method, the agar medium comprisesthe drug, pyrazinamide.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present inventors disclose herein a novel agar medium andmethods of using such a medium. The medium is generally referred toherein as HSTB agar medium. The agar medium is useful for the isolationof Mycobacterium tuberculosis from patient specimens, for thesub-cultivation of Mycobacterium tuberculosis pure cultures, and fordirect and indirect drug susceptibility testing against Mycobacteriumtuberculosis. The agar medium of the present invention has the followingadvantages over prior agar mediums for the culture of M. tuberculosis:(1) it an be used in regular incubators in the absence of supplementalCO₂, because the medium has an acidic pH of 6.0-6.25, and because ituses an animal serum instead of the OADC that was conventional in thistype of medium prior to the present invention; (2) it can be used fortesting susceptibility to pyrazinamide (PZA) because it has a lower pHthan conventional media (PZA requires lower pH), enabling the testing ofPZA in combination with other tuberculosis (TB) drugs on the same plate;and, (3) it is much less expensive than conventional agar mediums for M.tuberculosis because the conventional OADC supplement is replaced withan animal serum.

[0025] More particularly, the present inventors have demonstrated thatthe agar medium of the present invention, HSTB agar, supports growth ofM. tuberculosis in regular incubators, without supplemental CO₂ in theatmosphere. This feature makes this medium more acceptable than theconventional Middlebrook 7H10/7H11 agar in settings where installationof CO₂ incubators, as in many developing countries, may represent aproblem. Another advantage of the HSTB agar over the conventional7H10/7H11 agar is that it contains animal serum instead of OADC, whichmakes it easier to standardize and lower in cost. The present inventorshave introduced the use of HSTB in two settings: one is a bi-platecontaining plain and selective HSTB agar (i.e., HSTB agar in thepresence of antimicrobial agents for the isolation of M. tuberculosiscultures), another is a multi-segmented plate containing plain agar, aswell as an agar with various drugs for direct and indirectsusceptibility testing of M. tuberculosis-containing samples andpreviously isolated M. tuberculosis cultures. The present inventors havedemonstrated that HSTB was more efficient than 7H11 agar and all othermedia tested (L-J slant, BACTEC and MGIT broth) in recovery from sputumspecimens evaluated. Moreover, the present inventors have demonstratedthe ability to determine critical concentrations of TB drugs using theHSTB agar of the present invention. The concentrations could clearlyseparate resistant and susceptible strains, could distinguish low andhigh levels of isoniazid (INH) resistance, and could detect resistancein cultures containing various proportions of resistant bacteria—100%,50%, 10%, 1%. Results of an indirect test on HSTB and 7H10/7H11 agarwere reportable in three weeks for more than 80% strains, as compared toa turnaround time for a test in liquid MGIT medium of two weeks for 41%cultures and an additional 37% after the third week. The advantage ofthe test on agar medium is that it gives an actual proportion ofresistant bacteria in the isolate, is more sensitive in detecting smallproportions of resistant bacteria in the culture, and is applicable toboth first- and second-line drugs. Finally, the cost of three quadrantHSTB agar plates with 11 drugs (including PZA) is about two-fold lessthan 7H10 agar, and 10-15 fold less than MGIT tubes for a test with fouror five drugs only.

[0026] The present inventors have further shown that the HSTB agarmedium of the present invention can be used for susceptibility testingof M. tuberculosis isolates with pyrazinamide by an agar proportionmethod. This test is less expensive than the conventional testing withPZA in the liquid BACTEC system, the medium can be used in both anindirect test with previously isolated cultures and in a direct testmethod with acid fast bacilli (AFB)-positive sputum specimens, and thetotal turnaround time (from the moment the patient's specimen isobtained) can be as short as three weeks for at least 85% of theAFB-positive specimens.

[0027] Accordingly, one embodiment of the present invention relates toan agar medium, comprising: (a) an agar base suitable for growth ofMycobacterium tuberculosis; and, (b) animal serum at a concentration ofbetween about 8% and 12% of the final volume of the agar medium. Themedium is at a pH of between about 6.0 to about 6.25. As discussedabove, the novel agar medium of the present invention can also bereferred to herein as HSTB agar.

[0028] One essential component of the agar medium of the presentinvention is the agar base 16 of the medium. According to the presentinvention, an agar base that is suitable for the growth of Mycobacteriumtuberculosis can be any agar base that contains basic ingredients whichwill support the growth of Mycobacterium tuberculosis (pure cultures orspecimens containing Mycobacterium tuberculosis), when such base issupplemented with a suitable growth supplement (e.g., animal serum,according to the present invention, or OADC or ADC according toconventional recipes). Typically, the agar base is provided as a powderbase, and therefore is hydrated for use. To prepare the agar medium, theagar base is hydrated, supplemented with the growth supplement, andadjusted to the desired pH, for example by the addition of one or moresalts, such as monopotassium phosphate (KH₂PO₄). Additional ingredientscan be added as desired, such as glycerol as an additional nutritionalcomponent, although such ingredients are not considered to be essentialingredients of the agar medium. In addition, as discussed below, variousantimicrobial agents and tuberculosis drugs can be incorporated into themedium for the purpose of isolating M. tuberculosis cultures and fordrug susceptibility testing. Currently, there are two agar bases thatare particularly suitable for use in the agar medium of the presentinvention, Middlebrook and Cohn 7H10 agar (also referred to as “7H10agar” or “7H10”) and Middlebrook and Cohn 7H11 agar (also referred to as“7H11 agar” or “7H11”). 7H11 is 7H10 agar with the addition ofpancreatic digest of casein. It is available commercially throughseveral manufacturers (e.g., BBL). These media have been described indetail by Middlebrook and colleagues (e.g., Middlebrook et al., Amer. J.Publ. Health, 48:844-853, 1958; Cohn et al., Am. Rev. Respir. Dis.98:295-296, 1968) and represent the art standard agar bases for theculture of Mycobacterium tuberculosis. The powder base, 7H10 agar,contains agar, combination of seven salts, L-glutamic acid, pyridoxine,biotin, malachite green. It is available commercially from a number ofmanufacturers. It is to be understood that variations of theconventional 7H10/7H11 agar bases could be derived by one of skill inthe art, and it is contemplated that such agar bases could be used inthe agar medium of the present invention.

[0029] One essential feature of the agar medium of the present inventionis the pH of the medium. Prior to the present invention, theconventional agar media for M. tuberculosis were required to beformulated at a pH of 6.8, despite the knowledge that the optimal pH forM. tuberculosis is 6.2-6.4. Attempts to alter the conventional agarmedia to a pH of 5.5 by the substitution of ADC for the conventionalsupplement, OADC, resulted in very poor growth of the M. tuberculosis onthe medium. However, the first-line tuberculosis drug, pyrazinamide,only works at acidic pH and has not previously been successfully used inan agar medium for M. tuberculosis drug susceptibility testing. Thepresent inventors have demonstrated that the combination of an acidic pHwith the use of animal serum in the agar medium produces an agar mediumthat is more efficient than 7H10/7H11 agar and all other media tested(L-J slant, BACTEC and MGIT broth) in recovery of M. tuberculosis frompatient specimens, and allows for the first time the testing for PZAsusceptibility in an agar medium, alone or in conjunction with thetesting of other tuberculosis drugs. According to the present invention,the pH of the agar medium is between about pH 6.0 and about pH 6.25, andin another embodiment, between about pH 6.0 and pH 6.2, and in anotherembodiment, between about pH 6.1 and pH 6.2, and in another embodiment,is about pH 6.1. The use of the term “about” with regard to the pH ofthe medium refers can refer to a variance of up to 0.1 in eitherdirection (i.e., “about 6.0” can include a pH of 5.9 and 6.1). The pH ofthe medium is adjusted to the desired pH by any suitable method as iswell known in the art, including by the addition of one or more acids orbases to the medium, which are typically provided in the form of a salt.

[0030] Another essential feature of the agar medium of the presentinvention is the use of an animal serum at a concentration of from about8% to about 12% of the final volume of the medium. As discussed above,prior attempts to use an animal serum as a nutritional supplement inagar medium for M. tuberculosis failed because of inconsistent resultswith different serum batches. Therefore, a combination of standardizedproducts, sodium oleate with bovine albumin, was developed and used asthe major component of the OADC supplement. The role of the bovinealbumin is to neutralize the oleic acid, which is toxic to mycobacteria,in case of breakdown of sodium oleate. This composition required a finalpH of 6.8, which, as described above, is not optimal for the growth ofM. tuberculosis, and prevented the testing for susceptibility to thefirst-line drug, pyrazinamide. The present inventors have now discoveredthat the use of animal serum in place of the conventional OADCsupplement allows for the growth of M. tuberculosis on the agar mediumat a much lower pH, and further allows for the testing of tuberculosisdrugs in an agar setting which require acidic pH (e.g., PZA). Accordingto the present invention, the animal serum can be any animal serum, or asynthetic variant of an animal serum, that can readily be incorporatedinto a medium. Preferred animal sera include, but are not limited to,fetal bovine serum, calf bovine serum, bovine serum, fetal equine serum,and equine serum. As discussed above, the serum is added to the mediumat a final concentration of about 8% to about 12% of the final volume ofthe medium. Preferably, the serum is added at a final concentration offrom about 9% to about 11%, and more preferably, at about 10%. As usedwith regard to the concentration of serum in the medium, the term“about” can include a variance of 1% in either direction, even thoughthe target concentration is the stated concentration (e.g., “about 10%”can include 9% and 11%).

[0031] In one embodiment of the present invention, the HSTB agar canadditionally include one or more antimicrobial agents, which are addedto prevent the growth of contaminants on the medium, thereby enhancingthe usefulness of the medium for isolating cultures of Mycobacteriumtuberculosis. According to the present invention, HSTB agar thatcontains antimicrobial agents to prevent the growth of contaminants canbe referred to as “selective HSTB agar”. Typically, the selective HSTBis used to isolate M. tuberculosis cultures from a patient's specimen(e.g., a sputum specimen). Selective HSTB can also be used as one typeof control plate alongside a separate plate containing a tuberculosisdrug that is used for direct susceptibility testing of a sample from apatient. As used herein, the term “antimicrobial agent” is generallyintended to refer to any agent (i.e., a drug) that will inhibit thegrowth of microorganisms other than Mycobacterium tuberculosis, butwhich do not substantially inhibit the growth of the M. tuberculosis.Therefore, in the present invention, reference to an antimicrobial agentis generally not intended to refer to the tuberculosis drugs that areused in susceptibility testing. Antimicrobial agents for use in the agarmedium of the present invention include any agent that inhibits thegrowth of a microorganisms (including bacteria, fungi, viruses, andother mycobacteria) other than Mycobacterium tuberculosis, but which donot substantially inhibit the growth of the M. tuberculosis. Preferredantimicrobial agents for use in a selective agar medium of the presentinvention include, but are not limited to, polymixin B, carbenicillin,amphotericin B, and trimethoprim lactate. In one embodiment, theantimicrobial agents are present at a final concentration of: 200units/ml polymyxin B, 10 μg/ml amphotericin B, 50 μg/ml carbenicillin,and 20 μg/ml trimethoprim. It is to be understood, however, that it iswell within the ability of one of skill in the art to alter theseconcentrations as desired to inhibit the growth of contaminants on themedium. Typically, the antimicrobial agents are not used together in themedium with a tuberculosis drug for susceptibility testing. Instead, aseparate plate, segment of a plate or well containing the antimicrobialagent(s) is used alongside (in addition to) a plate, segment of a plateor well that contains the tuberculosis drug. An example of a bi-platecontaining plain HSTB medium on one half (one segment), and selectiveHSTB in the other half, is described below.

[0032] One embodiment of the present invention includes the HSTB agarmedium of the present invention (described above) which additionallycontains one or more drugs that are used to test Mycobacteriumtuberculosis for susceptibility to the drug. Such drugs can include anydrug that has been or will be identified as suitable for the inhibitionof growth or the destruction of Mycobacterium tuberculosis, and aretherefore potentially useful for the treatment of tuberculosis. Suchdrugs can also be referred to herein as “tuberculosis drugs”. Since somestrains of Mycobacterium tuberculosis are resistant to some tuberculosisdrugs, it is desirable to test a sample containing M. tuberculosis froma patient with tuberculosis against a variety of drugs, includingvarious doses of some drugs, in order to evaluate and select one or moredrugs and doses that will be best for use in the patient. Therefore, thepresent invention includes the incorporation of any tuberculosis druginto the agar medium of the present invention for the purpose of testinga sample of M. tuberculosis for susceptibility to the drug. Such drugsinclude, but are not limited to, isoniazid (INH), streptomycin sulfate(SM), di-hydro-streptomycin (SM), rifampin (RMP), pyrazinamide (PZA),ethambutol (EMB), etionamide (ETA), capreomycin sulfate (CM), amikacin(AK), kanamycin sulfate (KM), levofloxacin, p-aminosalicylic acid (PAS),D-cycloserine (CS), and clofazimine (CF).

[0033] The drugs to be tested for susceptibility are typically preparedas working stocks as described in detail in the exemplary protocolsbelow, and are added to the medium with the animal serum before the agarhas completely cooled and solidified. For the susceptibility testing isdesirable to have a control medium which does not contain any drugs(i.e., plain HSTB or drug-free HSTB), and in some cases, a plate forisolation and/or control (for direct testing procedures) that containsjust the antimicrobial drugs (i.e., selective HSTB). To provide the HSTBor selective HSTB, one option is to prepare additional plates containingthe various agar medium types to be used alongside the plates containingthe HSTB with the tuberculosis drugs. In a preferred embodiment,however, a single plate is segmented such that one portion of the platecan contain plain HSTB agar, and other portions of the plate can containselective HSTB agar or HSTB agar containing a tuberculosis drug. In thismanner, it is possible to prepare bi-plates (i.e., plates having twosegments, one with plain HSTB agar medium and one with selective ordrug-containing HSTB agar medium), as well as multi-segmented plates(i.e., plates having three, four, five, six, or more segments: one withplain HSTB agar, and each of the other segments with selective ordrug-containing HSTB agar medium, wherein each of the other segmentcontains a different tuberculosis drug, or a different dose of the sametuberculosis drug). Segmented plates are available commercially andcontain dividers within a single plate so that various types of mediumcan be poured into individual segments of the plate withoutcross-contaminating other segments. Alternatively, one can use a platecontaining several different wells, wherein each well is filled withagar medium of a different type. Such culture plates are also availablecommercially.

[0034] In one embodiment, the HSTB agar medium of the present inventioncontains one, two or a few different drugs, wherein each of the drugs isadded to a portion of the HSTB agar medium and then plated into aseparate plate, separate well, or separate segment of a segmented plateso that the drugs can be tested in one experiment against the sameinoculum. In one embodiment, the drugs are isoniazid or rifampin,wherein each of the drugs is isolated within a segment of the agarmedium on a single plate, or within agar medium contained in separatewells in a multi-well plate. In a preferred aspect of this embodiment,the isoniazid is present in two segments of the agar medium, whereineach segment (or well of a multi-well plate) contains a differentconcentration of isoniazid (e.g., a high dose and a low dose).Preferably, another segment or well contains drug-free HSTB agar medium.In another embodiment, the HSTB agar medium of the present inventioncontains the drug, pyrazinamide, wherein the pyrazinamide is isolatedwithin a segment of the agar medium on a single plate, and wherein theother segment of the medium is drug-free (plain agar). Any of thetuberculosis drugs disclosed herein are suitable for directsusceptibility testing, wherein a sample taken from a patient (e.g., asputum sample) is directly inoculated onto the agar medium (i.e.,without isolating the M. tuberculosis from the specimen first). It is tobe noted, however, that any of the drugs disclosed herein can also beused in indirect susceptibility testing (i.e., wherein the sample is aculture of M. tuberculosis that has previously been isolated from apatient sample, such as by culturing on a selective HSTB agar medium ofthe present invention).

[0035] In another embodiment, the HSTB agar medium of the presentinvention contains combinations of several different drugs, wherein eachof the drugs is added to a portion of the HSTB agar medium and thenplated into a separate plate, separate well, or separate segment of asegmented plate, so that the drugs can be tested in one experimentagainst the same inoculum. In one exemplary embodiment, the drugs to beincluded in a single segmented plate include: isoniazid, rifampin,pyrazinamide and either of streptomycin sulfate ordi-hydro-streptomycin, and wherein each of said drugs is isolated withina different segment of said agar medium. In another exemplaryembodiment, the drugs to be included in a single segmented plateinclude: ethambutol, etionamide, levofloxacin, capreomycin sulfate, andeither of amikacin or kanamycin sulfate, and wherein each of said drugsis isolated within a different segment of said agar medium. In yetanother exemplary embodiment, the drugs to be included in a singlesegmented plate include: p-aminosalicylic acid, D-cycloserine, andclofazimine, and wherein each of said drugs is isolated within adifferent segment of said agar medium. These combinations of drugs areparticularly suitable for indirect testing of patient samples (i.e., thepatient sample is first cultured, for example on selective HSTB, toisolate the M. tuberculosis, and is then inoculated onto thesusceptibility testing medium). It is to be understood that thesecombinations of drugs to be tested together are merely exemplary innature; it is well within the ability of one of skill in the art todesign different combinations of tuberculosis drugs for testing againsta patient sample. Therefore, the present invention contemplates the useof any one of the above-identified tuberculosis drugs, alone or incombination with any one or more of the others of the above-identifiedtuberculosis drugs or with any other tuberculosis drug that has been orwill be identified, in the agar medium of the present invention, for thepurpose of direct or indirect susceptibility testing. It is an advantageof the agar medium of the present invention that drugs that must be usedin an acidic environment (e.g., pyrazinamide) can be used in the samescreening with other drugs that do not have such requirements.

[0036] The amount of a given drug to incorporate into a given volume ofthe agar medium of the present invention can be readily determined bythose of skill in the art. This amount is typically determined byexperimentally determining the critical concentration of a given drug inthe HSTB medium, by measuring the highest MICs of the tuberculosis drugfor strains that are known to be susceptible to the drug, and the lowestMICs for clinical isolates that are known to be resistant to the drug.Ideally, the critical concentration of a drug is the concentration atwhich the majority of the drug-susceptible strains are inhibited, whilethe majority of the drug-resistant strains can grow. Based on thecritical concentration of drug in the medium, one can then determine thedrug-resistance growth level or breakpoint, to be used when screeningunknown cultures for drug susceptibility on the given drug (i.e., thepercentage of growth on the drug as compared to in the absence of thedrug, above which a culture is considered to be resistant to the drug).The determination of the critical concentrations of INH and RMP on HSTBis exemplified in Example 3. The determination of the criticalconcentration for PZA is exemplified in Example 5.

[0037] One embodiment of the present invention relates to a method forculturing Mycobacterium tuberculosis. The method comprises inoculatingan agar medium of the present invention with a sample containingMycobacterium tuberculosis. The agar medium can be the plain HSTB agarmedium of the present invention as described above, the selective HSTBmedium, or any tuberculosis drug-containing HSTB agar medium asdescribed above. Methods for the inoculation of an agar medium are wellknown in the art, and include smearing, pipetting or pouring a sampleonto the top of the agar medium and allowing the liquid in the sample toabsorb into the surface of the medium. The sample containing theMycobacterium tuberculosis can include any suitable sample, including anundiluted sample obtained directly from the patient (e.g., a sputumsample), a diluted sample obtained directly from the patient, or aculture of Mycobacterium tuberculosis that has been isolated previouslyfrom a patient sample. In one embodiment, sputum specimens are processedby the NaOH-NALC method with pH neutralization using a properconcentration procedure (described in detail below). If the specimen isto be diluted, the proper dilution can be determined by those of skillin the art. Standard dilutions include a two-fold dilution, a three-folddilution, a four-fold dilution, a five-fold dilution, a ten-folddilution, a one hundred-fold dilution, a one thousand-fold dilution, ora ten thousand-fold dilution.

[0038] Another embodiment of the present invention relates to a methodfor testing the drug susceptibility of a culture of Mycobacteriumtuberculosis. The method includes the steps of: (a) inoculating an agarmedium of the present invention with a sample containing Mycobacteriumtuberculosis, wherein the agar medium contains an amount of at least onedrug effective for selection against Mycobacterium tuberculosisorganisms that are susceptible to the drug; (b) incubating theinnoculated agar medium for a time sufficient to detect growth of theMycobacterium tuberculosis in the absence of a growth-inhibiting drug;and, (c) measuring growth of the Mycobacterium tuberculosis on the agarmedium as compared to growth of the Mycobacterium tuberculosis on theagar medium in the absence of the at least one drug. A growth rate ofthe Mycobacterium tuberculosis on the agar medium containing the atleast one drug that is less than a pre-established drug-resistance levelfor the at least one drug, when compared to the growth rate of theMycobacterium tuberculosis on the agar medium in the absence of the atleast one drug, indicates that the Mycobacterium tuberculosis issusceptible to the at least one drug.

[0039] The agar medium of the present invention and drugs containedtherein, including suitable formats for presenting the drugs forscreening, have been described in detail above. The types of samplessuitable for screening and the method of inoculation of the medium hasalso been described above. The step of incubating typically occurs at atemperature of from about 35-37° C., typically in the dark, and can beperformed in the absence of supplemental CO₂ (i.e., in normal airconditions). In one embodiment, the step of incubating occurs for atleast 2 weeks, and preferably about 3 weeks, and is typically from about3 weeks to about 6 weeks. An incubation of longer than 6 weeks istypically undesirable, since after 6 weeks, typically represents thelimit for interpretation of the drug susceptibility test results. In oneembodiment, the sample can be initially cultured in a liquid medium, forexample a Bactec vial, a MGIT tube, or a Redox tube, etc., which mayshorten the turnaround time of the laboratory report and increase theoverall rates of culture recovery. After recovery from the liquidmedium, the culture is plated on the agar medium of the presentinvention, which may result in shorter incubation times. Of course, theincubation period can be adjusted and monitored readily by those ofskill in the art.

[0040] The final step in this method includes measuring the growth ofthe Mycobacterium tuberculosis on the agar medium as compared to growthof the Mycobacterium tuberculosis on the agar medium in the absence ofthe at least one drug. Techniques for measuring the growth ofMycobacterium tuberculosis on the agar medium of the present inventioninclude, but are not limited to, counting colonies on the plate under amicroscope (e.g., a dissecting microscope). Typically, the number ofcolonies in a drug-containing segment or well is divided by the numberof colonies grown on the drug-free control segment or well andmultiplied by 100. If the percentage is greater than, or equal to, apre-established drug-resistance level for the particular drug, then theculture is considered to be resistant to that drug. If the percentage isless than the pre-established drug-resistance level, then the culture isconsidered to be susceptible to that drug.

[0041] A pre-established drug-resistance level is a level of growth of aculture which represents the “break point” between a culture beingresistant to the drug or susceptible to the drug. As described above, acritical concentration of a given drug can be determined for the drug inthe agar medium of the present invention (e.g., see Examples 3 and 5).The drug-resistance level of growth is established by determining apercentage of growth on the critical concentration of the drug, ascompared to the growth in the absence of the drug (i.e., 100%), belowwhich the culture is considered to be susceptible to the drug. Thedrug-resistance level, or breakpoint, for certain of the tuberculosisdrugs listed herein in HSTB agar medium has been determined by thepresent inventors, or can readily be determined, by those of skill inthe art. For all of the tuberculosis drugs disclosed herein, with theexception of pyrazinamide, the pre-established drug-resistance level ofgrowth is 1% (these are international standards). Therefore, for testingon these drugs, if the growth rate of the Mycobacterium tuberculosis onthe agar medium containing the drug is less than 1% of the growth rateof the Mycobacterium tuberculosis on the agar medium in the absence ofthe drug, the sample of Mycobacterium tuberculosis is susceptible to thedrug. The breakpoint for pyrazinamide is 10% (this is an internationalstandard). Therefore, for testing on pyrazinamide, if the growth rate ofthe Mycobacterium tuberculosis on the agar medium containing thepyrazinamide is less than 10% of the growth rate of the Mycobacteriumtuberculosis on the agar medium in the absence of pyrazinamide, then theMycobacterium tuberculosis is susceptible to pyrazinamide. Thebreakpoint, or pre-established drug-resistance growth rate, for otherdrugs can be determined by those of skill in the art using the guidanceprovided herein. If growth is present on the drug-containing media, thecolonies must be counted. Over-inoculation may result in a falseresistant interpretation.

[0042] In a preferred embodiment, a quality control strain ofMycobacterium tuberculosis which has previously been characterizedagainst the drugs to be tested is included on a duplicate set of platesso that the quality of the plates can be confirmed.

[0043] The following discussion provides further guidance on thepreparation and use of the HSTB agar medium of the present invention. Itis to be understood, however, that the protocols are to be considered tobe exemplary, and that one of skill in the art, given the discussionherein, will be able to make modifications to the protocols as desired.Such variations that result in an agar medium or method that fallswithin the scope of the present claims is considered to be part of thepresent invention.

[0044] General Design of Plates Using HSTB Agar Medium

[0045] In general, three different types of plates are of interest forthe isolation, sub-cultivation, and direct or indirect drugsusceptibility testing of Mycobacterium tuberculosis: (1) whole platescontaining plain HSTB agar (e.g., 20.0 ml per 100×15 nun plate); (2)bi-plates containing plain HSTB agar in one half of the plate (e.g.,10.0 ml) and selective HSTB agar in the second half; and (3) bi- ormulti-segment plates containing plain HSTB agar in one segment and HSTBagar containing a suitable amount of a drug to be tested in each of theother segments (i.e., with each different drug or each differentconcentration of the same drug being contained within a separatesegment).

[0046] Examples of Suitable Plate Designs

[0047] Examples of plates that are particularly useful for testing drugsusceptibility/resistance of M. tuberculosis include, but are notlimited to:

[0048] (1) Bi-plates for susceptibility testing with PZA, or any otherdrug as discussed previously herein, with side 1 of the plate containingHSTB agar with a suitable concentration of PZA (e.g., 1200 μg/ml), andside 2 containing the drug-free HSTB agar as control.

[0049] (2) Multi-Plate A—a four-segment plate for direct drugsusceptibility test with two drugs only (INH and RMP). It is advisableto use it along with a bi-plate containing plain and selective HSTBagar. The distribution of the segments or wells in the drug-containingplates is as follows: 1—Drug-free control; 2—0.2 μg/ml; 3—INH 1.0 μg/ml;and 4—RMP 1.0 μg/ml.

[0050] (3) Multi-Plate B—a six-well plate for indirect susceptibilitytesting with four drugs (INH, RMP, SM, and PZA). The distribution of thesegments or wells in the drug-containing plates is as follows:1—Drug-free control; 2—INH 0.2 μg/ml; 3—INH 1.0 μg/ml; 4—RMP 1.0 μg/ml;5—SM 8.0 μg/ml; 6—PZA 1200 μg/ml.

[0051] (4) Multi-Plate C—a six-well plate for an indirect test with fivemore drugs (EMB, ETA, AK or KM, CM, Levofloxacin). The distribution ofthe segments or wells in the drug-containing plates is as follows:1—Drug-free control; 2—EMB 14 μg/ml; 3—ETA 14 μg/ml; 4—AK (KM) 6.0μg/ml; 5—CM —10.0 μg/ml; 6—Levofloxacin 4.0 μg/ml.

[0052] (5) Multi-Plate D—a four-segment plate for an indirectsusceptibility test with three reserve drugs (PAS, cycloserine,clofazimine). The distribution of the segments or wells in thedrug-containing plates is as follows: 1—Drug-free control; 2—PAS 8.0μg/ml; 3—Cycloserine 60.0 μg/ml; 4—Clofazimine 6.0 μg/ml.

[0053] Protocol for Preparation of HSTB Agar

[0054] List of Supplies and Working Stocks:

[0055] Standard Laboratory Equipment:

[0056] Biosafety cabinet for sterile work (class I or II)

[0057] Autoclave

[0058] Regular incubator, set at 35-37° C.

[0059] Refrigerator and containers or brown paper bags to protect theplates from light

[0060] Analytical balance

[0061] Waterbath set at 54-56° C.

[0062] Vortex

[0063] 70° C. freezer

[0064] Magnetic stir plate

[0065] Supplies (Example Source):

[0066] Sterile four-segment plastic plates (Fisher)

[0067] Sterile plastic 6-well tissue culture plates (Life Sci. Prod.)

[0068] Plastic zip-lock bags (sandwich bags)

[0069] Sterile Erlenmeyer flasks with frosted mouth, 500 ml or 1000 ml

[0070] Sterile pipets, 1 ml, 5 ml, 10 ml, 25 ml

[0071] Pipetting device

[0072] Middlebrook and Cohn 7H10 agar base powder (Fisher)

[0073] Calf serum filter sterilized (Sigma)

[0074] Glycerol

[0075] KH₂PO₄ (Fisher)

[0076] Weighing dishes (Fisher)

[0077] Weighing spatula

[0078] De-ionized water

[0079] Sterile water for irrigation (Abbott)

[0080] 15 ml conical tubes (Fisher)

[0081] Graduated cylinder, 100 ml

[0082] Volumetric flasks, 25 ml and 50 ml

[0083] Magnetic stir bars

[0084] Antimicrobial Agents for Selective Medium

[0085] PACT: Drugs Used in the Selective Agar (if Bi-Plates are Preparedfor Culture Isolation to be Used along with Plates of Type A):

[0086] Polymixin B (Sigma, A3800406-001)

[0087] Add 5.0 ml of sterile distilled water to 50 mg to obtain 100,000units per ml. Dispense in 1.0-ml amounts and store at −70° C.

[0088] Carbenicillin (Geopen) (Sigma, C1389)

[0089] Add 50 ml of sterile distilled water to 5 g to obtain a solutioncontaining 100,000 μg/ml. Dispense in 1.0-ml amounts and store at −70°C.

[0090] Amphotericin B (Fungizone) (Sigma, A4888)

[0091] Add sterile distilled water to a vial containing the drug toobtain a solution of 10,000 μg/ml. Dispense in 1.0-ml amounts and storeat −70° C. This drug is light sensitive. Keep vials wrapped in foil.

[0092] Trimethoprim Lactate (Sigma, T7883)

[0093] Activity is 1000 μg/ml. Prepare an aqueous solution containing10,000 μg/ml. Filter sterilize and store at −70° C.

[0094] Preparation of Drugs for Susceptibility Testing (Example Source)

[0095] Drugs must be chemically pure, not from the pharmacy stock. Whenordering drugs, one should get information regarding the biologicalactivity in micrograms activity per milligram weight. If the biologicalactivity is not 100% of the dry weight, the following approach is usedto prepare the stock solution. To determine the needed powder weight,multiply the desired drug concentration (μg/ml) by the desired volume ofthe solution to be made (ml), and divide by the drug potency (mg pergram). For example, to make 25 ml of a solution containing 10,000 μg/mlof a drug with 800 mg per gram potency: [10,000×25]:800=312.5 mg.

[0096] This amount of the drug powder should be weighed on a wellcalibrated analytical balance, and fully dissolved in 25 ml ofappropriate solvent, using volumetric class A flasks.

[0097] Stock drug solutions of the water-soluble TB agents (all exceptRMP) are made with sterile or non-sterile water but must befilter-sterilized in either instance using a membrane filter with a poresize of 0.22 μm. Subsequent dilutions are made in sterile water forirrigation. Stock solutions should be made at concentrations of 1000μg/ml to 10,000 μg/ml, as shown below, in a volumetric flask and storedin sterile freezer vials at −70° C. for up to 6 months. After thawing,the drug must be used immediately. The drug solution should never bere-frozen.

[0098] Most of the pure drug powders listed below are also availablefrom US Pharmacopean Convention, Inc., Reference Standards, OrderDepartment, 12601 Twinbrook Parkway, Rockwille, Md. 20852.

[0099] Isoniazid (INH, Sigma)

[0100] Usual activity is 1000 μg/mg. Prepare an aqueous solutioncontaining 2,000 μg/ml and filter sterilize. Dilute in sterile water to20 μg/ml and 4 μg/ml for the working solutions.

[0101] Streptomycin Sulfate or Di-Hydro-Streptomycin (SM, Sigma, S6501)

[0102] Usual activity is 780 μg/mg. Prepare an aqueous solutioncontaining 8, 000 μg/ml and filter sterilize. Dilute in sterile water to160 μg/ml for the working solution.

[0103] Rifampin (RMP, Sigma)

[0104] Activity is 1000 μg/mg. Prepare a solution containing 2,000 μg/mlin methanol or in 95% ethanol. This is a self-sterilizing solution.Dilute to 20 μg/ml in sterile water for the working solution.

[0105] Pyrazinamide (PZA, Sigma)

[0106] Usual activity is 1000 μg/mg. Prepare an aqueous solutioncontaining 12,000 μg/ml. Filter sterilize. This is the working solution.

[0107] Ethambutol (EMB, Sigma)

[0108] Usual activity is 1000 μg/mg. Dissolve 200 mg in 20 ml ofdistilled water to have a solution of 10,000 μg/ml. Filter sterilize.

[0109] Etionamide (ETA, Sigma)

[0110] Usual activity is 10,000 μg/mg. Dissolve 100 mg in 20.0 ml ofethylene glycol (analytical grade) or 250 mg in 50.0 ml to obtain astock solution containing 5,000 μg/ml. Incubate overnight at 37° C. forself-sterilization. Heat gently if not completely dissolved. Aliquots(1.5 ml) should be kept at −20° for not more than 3 months.

[0111] Capreomycin Sulfate (CM, Sigma)

[0112] Activity varies with each lot. Taking into account the actualactivity, prepare an aqueous solution containing 10,000 μg/ml. Filtersterilize, and keep the aliquots for not more than 2 months at −20° C.

[0113] Amikacin (AK, Sigma, Free Base) or Kanamycin Sulfate (KM, Sigma)

[0114] Activity is varied. Prepare aqueous solution containing 10,000μg/ml of the active product, filter sterilize, and keep the frozenaliquots for not more than 6 months.

[0115] Levofloxacin (from RW Johnson or Ortho-McNeil)

[0116] Activity is 1,000 μg/mg. Prepare aqueous solution of 80.0 μg/ml,filter sterilize. This is a working solution.

[0117] p-aminosalicylic Acid (PAS, Sigma)

[0118] Prepare a solution of 8,000 μg/ml taking into account the actualpotency of the batch.

[0119] D-Cycloserine (CS, Sigma)

[0120] Usual activity is 1,000 μg/mg, but in case of DL-Cycloserine theusual activity is 500 μg/mg. Prepare 6,000 μg/ml solution in a Na₂CO₃solution that has pH 10.0. The later is prepared by using 0.1% solutionof Na₂CO₃ to be added to 100 ml of distilled water until pH reaches10.0. Frozen aliquots should be kept for not more than one month.

[0121] Clofazimine (CF, Sigma)

[0122] Usual activity is 1,000 μg/mg. A 100 μg/ml solution should bemade in DMSO.

[0123] Subsequent dilutions are made also in DMSO. The aliquots can bekept for not more than one month at room temperature, protected fromlight.

[0124] Protocol for Production of Plain HSTB Agar for any of theDrug-Free Controls, for a Bi-Plate, or for a Whole Plate:

[0125] The optimal volume that is easy to handle in the clinicallaboratory is 200 ml or 300 ml agar per 500-ml or 1000-ml flask,although the volumes can be adjusted, as can be readily determined byone of skill in the art. Calculations for drug-containing media aregiven here for 200 ml of the medium to prepare 40 drug-plates, based onapproximately 5 ml of agar per well or segment. One of skill in the artwill be able to modify the calculations to accommodate different volumesof medium. In addition, one of skill in the art will be able to modifythe percentage of animal serum, or modify the pH, within the ranges setforth previously herein, as desired. The following recipes are designedto produce an HSTB medium containing about 10% animal serum and to be ata pH of from about 6.0 to about 6.25.

[0126] Plain HSTB Agar

[0127] In a 500 ml Erlenmeyer flask add the following:

[0128] Magnetic stir bar

[0129] 3.6 g 7H10 agar base

[0130] 1.2 g KH₂PO₄

[0131] 180 ml de-ionized water

[0132] 1 ml glycerol

[0133] Autoclave at 121° C. for 10-12 min.

[0134] Cool in waterbath to 54-56° C.

[0135] Add 20 ml of sterile calf bovine serum (CBS), or other suitableanimal serum as described previously herein, warmed to room temperature.Stir ingredients using a magnetic stir bar on a magnetic stir platebeing careful not to create bubbles. Pour approximately 5 ml of agarinto the segments or wells by tilting the flask, or use a pipet. Alwaysuse aseptic technique in all media preparation procedures. Allow theplates to completely cool and solidify. Store plates in plastic bags (tokeep from drying out) away from light at 4-5° C.

[0136] If the bi-plate for culture isolation is prepared, the procedure,is the same, but 10 ml is used per each half of the plate.

[0137] Selective Agar

[0138] This medium is intended for the bi-plates if they are used alongwith plates of type A (or other plates designed for direct drugsusceptibility testing). Preparation of the medium is the same as forthe plain agar (see above), but along with the serum, four drugs (PACT)are added to have the final concentrations shown below. About 10 ml ofthis medium is used in the second half of the bi-plate. Polymyxin B* 200units/ml (25 μg/ml) Amphotericin B  10 μg/ml Carbenicillin  50 μg/mlTrimethoprim  20 μg/ml

[0139] Agar with INH Low Concentration

[0140] Follow the directions for the plain agar except reduce the waterto 170 ml. After the agar has cooled to 54-56° C. add 20 ml of serum and10 ml of the INH working solution (4.0 μg/ml) for a final concentrationof 0.2 μg/ml.

[0141] Agar with INH High Concentration

[0142] Follow the directions for the plain agar except reduce the waterto 170 ml. After the agar has cooled to 54-56° C. add 20 ml of serum and10 ml of the INH working solution (20 μg/ml) for a final concentrationof 1.0 μg/ml.

[0143] Agar with RMP

[0144] Follow the directions for the plain agar except reduce the waterto 170 ml. After the agar has cooled to 54-56° C. add 20 ml of serum and10 ml of the RMP working solution (20 μg/ml) for a final concentrationof 1.0 μg/ml.

[0145] Agar with SM

[0146] Follow the directions for the plain agar except reduce the waterto 170 ml. After the agar has cooled to 54-56° C. add 20 ml of serum and10 ml of the SM working solution (160 μg/ml) for a final concentrationof 8.0 μg/ml.

[0147] Agar with PZA

[0148] Follow the directions for the plain agar except reduce the waterto 160 ml. After the agar has cooled to 54-56° C. add 20 ml of serum and20 ml of the PZA working solution (12,000 μg/ml) for a finalconcentration of 1200 μg/ml. Since this concentration is closed to thesaturation point, the thawed solution must be inspected for crystalsbefore using. Vortex and incubate for 10-15 minutes at 37° C. ifcrystals are present.

[0149] Agar with EMB

[0150] Prepare a working solution by diluting the stock solution 1:40 indistilled water. Prepare the agar in 170 ml, and add, after cooling, 20ml of serum and 10 ml of the working drug solution (280 μg/ml) to havethe final drug-concentration of 14 μg/ml.

[0151] Agar with ETA

[0152] Follow the same instruction as for EMB.

[0153] Agar with AK (KM)

[0154] Prepare a working solution by diluting 1:100 the stock solutionin water. Prepare agar in 168 ml, and add, after cooling, 20 ml of serumand 12 ml of the working drug solution (100 μg/ml) to have the finaldrug-concentration of 6.0 μg/ml.

[0155] Agar with CM

[0156] Prepare a working solution by diluting 1:100 the stock solution.Add to 160 ml of the cooled agar 20 ml of serum and 20 ml of the drugworking solution (100 μg/ml) to have the final drug-concentration of10.0 μg/ml.

[0157] Agar with Levofloxacin

[0158] Add 20 ml of serum and 10 ml of the stored (see above) drugworking solution (80 μg/ml) to 170 ml of cooled agar to have the finaldrug-concentration of 4.0 μg/ml.

[0159] Agar with PAS.

[0160] Prepare a working solution by diluting 1:100 the stock. Add 20 mlof serum and 20 ml of the working solution (80 μg/ml) to 160 ml ofcooled agar to have the final drug-concentration of 8.0 μg/ml.

[0161] Agar with CS.

[0162] Prepare a working solution by diluting the stock 1:10. Add 20 mlof serum and 20 ml of the working solution (60 μg/ml) to 160 ml ofcooled agar to have the final drug-concentration of 6.0 μg/ml.

[0163] Agar with CF.

[0164] To 168 ml of cooled agar add 20 ml of serum and 12 ml of theworking solution (100 μg/ml in DMSO—see above) to have the finaldrug-concentration of 6.0 μg/ml.

[0165] Composition of the Plates

[0166] The optimal is 40 plates per batch, although it is well withinthe ability of those of skill in the art to vary the batch according topreference. For example, six flasks of medium are needed for preparationof the six-well plates of type B or C, or four flasks for four-segmentplates of type A, at 200 ml each. The flasks should be madesimultaneously for such a batch. Each plate is labeled with the batchnumber or date of preparation and placed in an individual plastic bag.Each batch is stored in a container to be protected from light and keptrefrigerated until use.

[0167] Quality Control

[0168] A pre-characterized susceptible M. tuberculosis isolate should beused to ensure the potency of the drugs incorporated into the agar. Thisquality control strain should be inoculated onto the plates every time abatch of clinical isolates is tested. Plates should not be used untilproven appropriate in a test with the QC strain.

[0169] General Directions for Plate Preparation, Storage and Use

[0170] The drugs are incorporated into the medium in tentative criticalconcentrations such as those described above for the exemplary plates,but it is to be understood that these concentrations can be validated oraltered subsequent to further study. INH is included in twoconcentrations with the intention of detecting two types ofINH-resistance (low and high). Each plate is enclosed in a plasticzip-lock bag, and should be kept refrigerated until use. The tentativeshelf-life is three months from the date of preparation, which may beextended after proper evaluation. The plates should be kept out of lightduring storage and the subsequent use.

[0171] The plates should be removed from the refrigerator a few hoursbefore intended use, and placed at room temperature on the bench, inplastic bags, covered. The plates should be removed from the plasticbags prior to inoculation under the hood (biosafety cabinet), andlabeled appropriately. After inoculation, the plates should remain underthe hood until the inoculum is well absorbed by the medium. Sometimes,it is necessary to place the lid slightly off the plate to facilitatethis drying process. After the plates have dried, place them inside theplastic zip-lock bags, seal individually, and only after that removethem from the hood.

[0172] General Protocol for Drug Susceptibility Testing Using HSTB AgarMedium

[0173] The novel agar medium of the present invention (HSTB) is designedfor cultivation of M. tuberculosis in regular incubators, withoutsupplemental CO₂ in the atmosphere. By way of example, the followingprotocols describe the use of plates of type A, B and C, as discussedabove.

[0174] Culture Isolation and Direct Drug Susceptibility Test Using TypeA Plates:

[0175] The type A plate should be used for culture isolation and directsusceptibility testing for smear-positive specimens only. Sputumspecimens should be processed by the NaOH-NALC method with pHneutralization using a proper concentration procedure (see below). Atleast one more unit of culture medium, an L-J slant, or better, an agarbi-plate (with plain and selective HSTB agar), should be used forculture isolation. In addition, inclusion of a liquid medium, forexample a Bactec vial, a MGIT tube, or a Redox tube, etc, may shortenthe turnaround time of the laboratory report and increase the overallrates of culture recovery. All these culture media should be inoculatedwith an undiluted specimen. At the same time, to obtain valid results ofthe direct drug susceptibility test, the inoculum for plate A should bediluted, if necessary, based on the smear-examination results:

[0176] 1) specimen showing ≦25 AFB/field (1+)—inoculate 0.1 ml of theundiluted specimen per segment, or 0.2 ml if there is only ≦5 AFB/field;

[0177] 2) specimen with 25-50 AFB/field (2+)—inoculate one plate with0.1 ml of the undiluted specimen per segment and the other with 0.1 mlof a {fraction (1/10)} dilution (10⁻¹);

[0178] 3) specimen with 50-250 AFB/field (3+)—use undiluted specimen and10⁻² dilution;

[0179] 4) specimen with >250 AFB/field (4+)—use 10⁻² and 10⁻⁴ dilutions.

[0180] Indirect Drug Susceptibility Test Using Plates Type B, C or D

[0181] A bacterial suspension from colonies homogenized in a tubecontaining 7H9 broth and glass beads, or a culture grown in any of theliquid media, is adjusted to the optical density of a McFarland No. 1standard. Two dilutions are made at 10⁻² and 10⁻⁴ in saline or 7H9broth, inoculated in the amount of 0.1 ml per well, using two plates perculture. If a culture is obtained from a BACTEC vial with daily GI 999,dilute 10⁻¹ and 10⁻³ and inoculate 0.1 ml each per well. The coveredplates are tilted to spread the inoculum avoiding the edges as much aspossible and left under the hood for at least one hour until the liquidhas fully absorbed into the agar.

[0182] Incubation for All Plate Types

[0183] The bagged plates are removed from the safety cabinet, stacked nomore than six plates high, agar side down, in wire baskets, andincubated in regular incubators at 35-37° C for 21 days, protected fromlight. After incubation, upon removal from the incubator, the platesshould be turned up side down (agar up), and left in bags overnight atroom temperature to eliminate condensation. Some strains may not producevisible growth after 3 weeks of incubation. If the culture has not grownyet, the plates should be re-incubated for additional 3 weeks, but thereis a limit for interpretation of the drug susceptibility test results ifthe culture had to be incubated for 6 weeks (see below).

[0184] Counting of the Colonies and Interpretation

[0185] The results of the drug susceptibility test are considered validif the results with the QC strain(s) are in agreement with theestablished laboratory standards, and if the number of colonies grown indrug-free wells are no less than 50, but, even better, greater than 100.This number should not be greater than 300, especially forinterpretation of “resistant” results. The colonies are examined forpurity and counted under a dissecting microscope without removing theplate from the plastic bag. The results are reported as a percentage ofresistance. For this purpose, the number of colonies in adrug-containing well or segment is divided by the number of coloniesgrown on the drug-free control and multiplied by 100. If the percentageis greater than or equal to one, the culture is considered resistant tothat drug concentration for INH, SM, and RMP. The breakpoint for PZA is10%. If growth is present on the drug-containing media, the coloniesmust be counted. Over-inoculation may result in a false resistantinterpretation. The standard procedure is applicable for culturessufficiently grown after 3 weeks of incubation. For cultures not grownat 3 weeks and examined only after 6 weeks of incubation, the results ofthe drug susceptibility test are reportable only if the isolate shows nodrug resistance (no growth on the drug-containing medium).

[0186] The records during the period of evaluation of new agar platesshould include the following information: source of the inoculum(medium), its actual preparation, number of colonies grown in each well,and the interpretation. It is desirable that the laboratory'sconventional susceptibility method is performed along with the use ofthe new agar plates.

[0187] Biosafety

[0188] Specimen and/or culture handling including all procedural stepsup to the incubation step or removal of the plate from the plastic bagshould be performed in a Class 2 Biological Safety Cabinet in aBiosafety Level 3 facility. Use extra care with pipets or pipettingdevices that can result in high-pressure inoculation as the inoculum cansplatter off the agar medium and create an aerosol. Examination of theplates in an open bench area of the laboratory should be done withoutopening the plastic bags, as described above. Special attention shouldbe given to elimination of the condensation by placing the plates afterincubation and before examination in an upside down (agar up) positionovernight at room temperature. Removal of the plates from the plasticbags can be considered for the purpose of culture isolation fromcolonies grown on drug-free segments only. This work should be done inthe biosafety level 3 facility with all appropriate precautions.Cultures on agar plates should never be shipped through the mail due thepossibility of the agar shaking loose of the plate.

[0189] Preparation of Samples for Drug Susceptibility Testing

[0190] NaOH-NALC Digestion-Decontamination Procedure with Neutralization

[0191] Rationale. The problem faced in the laboratory is one ofbalancing the need for a gentle decontamination procedure that maintainsviability of the mycobacteria with the need to eliminate all otherorganisms. None of the decontamination techniques available meets thiscriterion perfectly. It should be realized that even under the best ofconditions only 10-20% of mycobacteria found in a clinical specimensurvive the decontamination process. The procedure outlined below(NaOH-NALC method with neutralization) exploits the relative resistanceof acid-fast bacteria to the effects of alkali and/or acids in order toseparate them from other microorganisms. The mucolytic agentN-acetyl-L-cysteine (NALC) is used for digestion of mucus to homogenizethe sputum. Sodium hydroxide (NaOH) is used to eliminate contaminatingmicroorganisms, while leaving an adequate number of viable mycobacteria.After decontamination, a phosphate buffer solution and a solution ofhydrochloric acid (HCl) should be used to neutralize the NaOH. Only thistechnique is suitable for inoculation of the agar, particularly for adirect drug susceptibility test on HSTB agar plates of type A.

[0192] Solutions Required

[0193] NaOH-NALC Solution (“Digestant” Solution)

[0194] Sodium hydroxide stock solution: dissolve 200 ml of NaOH solution(50% w/w) in 1000 ml distilled water.

[0195] Sodium citrate stock solution: dissolve 147 g of Sodium Citrate(Na₃C₆H₅O7.2H₂O) in 1000 ml distilled water.

[0196] Working solution: mix together the two solutions above and adddistilled water to make a final volume of 5000 ml. Store in brownplastic bottles, 250 or 500 ml in each, at 2-8° C., for not more than 2months.

[0197] Final digestant: Just before use, add 0.5 g ofN-acetyl-L-cysteine powder (NALC) to every 100 ml of the workingsolution. Prepare only the volume of final digestant needed for one day,because the mucolytic activity of NALC is lost upon standing. The finaldigestant must be used within 18-24 hours. The composition of the finaldigestant solution is:

[0198] 2% (0.5 M) NaOH

[0199] 0.1 M Sodium Citrate

[0200] 0.5% NALC.

[0201] To avoid a possibility of cross-contamination, never pour thedigestant solution (as well as other reagents) to the tubes containingspecimens directly from the flask. Instead, make aliquots of thissolution first, in volumes corresponding to the volumes of the specimens(8.0 ml each for this study), label with the volume and specimens'numbers, and place them against the corresponding specimens. Pouraliquots into corresponding specimens.

[0202] Phosphate Buffer Solution

[0203] To make a 0.5 M phosphate buffer solution with pH 6.0, dissolvein 1000 ml distilled water:

[0204] 8.7 g Na₂HPO₄ (dibasic sodium phosphate)

[0205] 59.7 g KH₂PO₄ (monobasic potassium phosphate)

[0206] 40 mg Phenol Red Sodium Salt

[0207] Note: these figures are for anhydrous chemicals. If phosphatesare hydrated, values must be adjusted accordingly.

[0208] If necessary, the solution may be warmed slightly to facilitatedissolving of the salts. Sterilize buffer by autoclaving for 15 min,label and store at 2-8° C. Aliquot buffer for each specimen, usinganother 50 ml centrifuge tube, then pour into each specimen rather thanpouring from a common flask. Make in advance aliquots, 16 ml each. Keepthem refrigerated.

[0209] Hydrochloric Acid

[0210] Hydrochloric acid (HCl), at a concentration of 0.5 M, will beneeded for neutralization of the processed specimens that have alkalinepH because of treatment with NaOH. The concentration of bulk“concentrated” HCl varies from about 36.538%. A 37% solution of HCl isequivalent to 11.9 M. To make 1000 ml of a 0.5 M HCl solution from 37%solution, first place about 800-900 ml of distilled water into avolumetric flask. Then add 42 ml of 37% solution, mix, and bring thetotal volume up to 1000 ml. When working with concentrated acids, forsafety's sake, always add the acid to a larger volume of water. NEVERadd water to concentrated acids because the generated heat can cause theacid to splash or explode. Make aliquots, 8.0 ml each, and keep themrefrigerated.

[0211] 20% Animal Serum Solution

[0212] Prepare a solution by adding 20 ml of sterile animal serum to 80ml of sterile distilled water, filter sterilize, and make 2.0-mlaliquots to be kept refrigerated. Two ml of this solution will be usedto re-suspend each pellet of bacteria after the decontaminationprocedure.

[0213] Procedure for Sputum Specimens Processing

[0214] 1. Sputum specimens are usually collected in sterile 50-mlplastic screw-cap centrifuge tubes. Specimens collected otherwise shouldbe transferred into such 50-ml tubes. Place 8 ml of sputum specimen intoa sterile 50-ml tube. If the submitted specimen contains more than 8 mlof sputum, the specimen should be distributed among 2 or 3 tubes. If thevolume of the submitted specimen is less than 8 ml, add, usingindividual pipettes for each specimen, sterile saline to bring thevolume up to 8 ml. Add 8 ml of the described above digestant solutionfrom the prepared aliquots to the specimen. The total volume of thespecimen is now 16 ml, and the concentration of NaOH in thesputum/digestant mixture is 1%, or 0.25 M. Stopper tightly and mix forapproximately 20 seconds on a vortex mixer. Do not swirl contents upinto the cap or shake by hand after vortexing.

[0215] 2. Allow the mixture to stand for exactly 15 minutes at roomtemperature.

[0216] 3. Add 16 ml of 0.5M phosphate buffer to the sputum/digestantmixture. Stopper tightly and mix carefully.

[0217] 4. Add 8 ml of 0.5M hydrochloric acid solution to the specimen.Stopper tightly and mix carefully. The pH of the specimen should now be7.0 or less, and the color of the specimen should now have a yellowcolor.

[0218] 5. Centrifuge at 3500×g for at least 25 minutes in a refrigeratedcentrifuge that has aerosol-containment buckets.

[0219] 6. Cautiously, avoiding disturbance of the pellet, decantsupernatant fluid into a canister/funnel arrangement containing 5%amphyl solution.

[0220] 7. Prepare smears by dipping a wooden applicator stick into theconcentrate and drawing it across the surface of the slide. Allow slidesto dry under the hood. Place on a slide warmer to heat-fix at 68° C. for2 hours.

[0221] 8. Stain slides, examine, and report results accordingly.

[0222] 9. Add 2.0 ml of sterile animal serum solution to the pellet.

[0223] 10. Inoculate various media for culture isolation with theundiluted concentrated specimen, and prepare dilutions for the directsusceptibility test on the plate A according to smear results.

[0224] The following examples are provided for the purpose ofillustration and are not intended to limit the scope of the presentinvention.

EXAMPLES Example 1

[0225] The following example describes cultivation of Mycobacteriumtuberculosis on HSTB agar medium without CO₂.

[0226] HSTB agar. The commercially available Middlebrook/Cohn 7H10 agarbase (BBL, Becton Dickinson and Co., Cockeysville, Md.) was dissolved indeionized water, 3.6 g of the powder per 160 ml of distilled water, in500-ml Erlenmeyer flasks containing magnetic stir bars. Then 1.2 g ofmonopotassium phosphate (KH₂PO₄) to acidify the medium and 1.0 ml ofglycerol was added per flask. After being autoclaved at 121° C. for 12min., the flasks were placed into the water bath to cool down to 54° C.The standard animal serum product to be added to the agar was obtainedfrom Sigma Chemical Co. The serum used was fetal bovine serum (FBS),calf bovine serum (CBS), or fetal equine serum (FES). Preliminarystudies indicated that any type of serum from these sources equallysupported growth of M. tuberculosis strains. Most of the resultspresented this and subsequent examples were performed with HSTB agarcontaining CBS in the amount of 10% to the final volume of the agarmedium. The most convenient way to prepare the agar in the clinicallaboratory setting is by making 200-ml portions of the medium. For thispurpose, flasks with agar cooled to 54° C., and 160 ml in each wereplaced as described above on the magnetic plate for subsequent mixturewith the added serum. Each 160-ml portion was supplemented with 40 ml ofa separately prepared mixture of 20 ml of serum (warmed up to the roomtemperature) plus 20 ml of a drug solution (See Examples 3-4), or 20 mlof distilled water for a drug-free medium (this Example), so that thefinal volume in each flask was 200 ml, and the final pH of the mediumwas 6.15±0.1. The same batches of agar were used in parallel to preparestandard 7H10 and 7H11 agar containing OADC for comparison with the HSTBagar.

[0227] Results. A total of 60 M. tuberculosis cultures were tested byincubation at 37° C. without supplemental CO₂, as well as in thepresence of 5-7% of CO₂ in the atmosphere. Growth of all strains wasrecovered on HSTB agar with or without CO₂ within three weeks ofcultivation. Only 52 of these strains were also tested for growth on7H10 and 7H11 agar plates. Four of these 52 strains did not grow onthese media without CO₂. The present inventors' have previouslydetermined that generally, about 20% of M. tuberculosis isolates willnot grow on 7H10/7H11 agar without 5-7% of CO₂ in the atmosphere. Themorphology of the colonies on HSTB agar was slightly different from thaton 7H10/7H11 agar: the colonies were smaller, more domed, and lessspread on the surface of the medium.

Example 2

[0228] The following example describes the comparison of ADC, OADC, andanimal serum as supplements in 7H10/7H11 agar media.

[0229] It was previously speculated that oleic acid may inhibit thebacterial growth at low pHs, and therefore it was suggested to use ADCinstead of OADC for 7H10/7H11 agar media to obviate the growthinhibition at the acidic pH of 5.5 (Butler et al., J. Clin. Microbiol.,16:1106-1109, 1982; Butler et al., Antimicrob Agents Chemother.,24:600-601, 1983). In an attempt to create optimal conditions for M.tuberculosis growth at the acidic pH, the present inventors comparedgrowth of three strains (H37Rv, Erdman and Atencio) on 7H11 agarsupplemented with 10% of OADS vs ADS vs equine fetal serum (EFS) vsbovine fetal serum (BFS), all obtained from Sigma.

[0230] The commercially available Middlebrook 7H10 agar base (BBL,Becton Dickinson and Co., Lockeysville, Md.) was dissolved in deionizedwater, 14.4 g per 660 ml. Then 4.7 g of monopotassium phosphate (KH₂PO₄)was added to acidify the medium. In addition, 0.72 g of caseinhydrolysate and 4.0 ml of glycerol were added. After autoclaving at 121°C. for 12 min, the medium was split into four sterile flasks, 160 ml pereach. The flasks were placed into the water bath to cool down to 54° C.After that, 20 ml of the animal serum (bovine or equine fetal or calfserum from Sigma Chemical Co.) was added up to the total volume of 200ml per flask. The final concentration of the serum was 10%, and thefinal pH was 6.15±0.1. For the purpose of comparison, similar media weremade with OADC or ADC instead of the animal serum.

[0231] Two sets of whole plates (to have a duplicate) of the agarcontaining each of the supplements, one having standard pH 6.8 and onewith pH 6.0, were inoculated simultaneously with 0.5 ml of the bacterialsuspension, to have approximately 100 to 200 CFU per plate by using a10⁻⁶ dilution of a bacterial suspension adjusted to the optical densityof the McFarland Standard No. 1.

[0232] The results of experiments with three strains are shown inTable 1. These preliminary data showed that the recovery of M.tuberculosis an agar media supplemented with BFS was equal and sometimeseven better than on the standard OADC-containing medium at pH 6.0, andno worse than on the media with pH 6.8. At the same time, growth at pH6.0 on the media supplemented with ADC was partially suppressed comparedwith the media with either OADC or BFS. In addition, we should stressthat the size and appearance of colonies at the three-weeks reading onthe Ph 6.0 medium with BFS was no different from that with pH 6.8. TABLE1 Number of CFU grown on four types of agar medium, each at pH 6.8 and6.0. Two counts separated by a slash represent the numbers on two platespH 6.8 pH 6.0 Supplement H37Rv Erdman Atencio H37Rv Erdman Atencio OADC56/32 — 70/29 72/71 29/13 76/77 OADC 50/63 77/68  82/102 — — — ADC 45/3173/60 100/101 5/6 3/9 39/72 EFS 55/68 57/91 96/92 57/66 52/41 0/0 BFS50/63 64/72 55/13 65/62 60/66 84/94

Example 3

[0233] The following example describes the determination of the INH andRMP critical concentrations for use in HSTB agar medium.

[0234] The purpose of this study was to determine the highest MICs ofthe tuberculosis drugs, isoniazid (NH) and rifampin (RMP) forpansusceptible strains, and the lowest MICs for INH- and RMP-resistantclinical isolates, using HSTB and 7H11 agar medium in parallel. A broadrange of concentrations of either INH or RMP was incorporated into HSTBand 7H11 agar media.

[0235] Preparation of the four-segment plates with two drugs.Four-segment plastic plates, 100×15 mm, were used to have plain agar(segment I), agar with 0.1 μg/ml and 1.0 μg/ml of isoniazid (segments IIand III), and agar with 2.0 μg/ml of rifampin (segment IV), plated atabout 5.0 ml of the medium per segment. The HSTB agar was prepared asdescribed in Example 1. Stock solutions of isoniazid (NH) were made indistilled water (filter-sterilized), and the stock solution of rifampin(RMP) was made in methanol. Aliquots of the stock-solutions werepreserved at −70° C. To make an agar containing 0.1 μg/ml of INH, 20 mlof the NH working solution containing 2.0 μg/ml of the drug was mixedwith 20 ml of serum, and added to 160 ml of agar cooled to 54° C. Toprepare the medium containing 1.0 μg/ml of INH, 20 ml of the workingdrug solution containing 10 μg/ml was mixed with 20 ml of serum, andadded to 160 ml of agar cooled to 54° C. For the RMP-containing agar, 20ml of the working aqueous solution containing 20 μg/ml of this drug wasmixed with 20 ml of serum, and added to 160 ml of agar, resulting in thefinal concentration of 2.0 μg/ml of RMP.

[0236] Results. The MICs of INH for 13 M. tuberculosis pan-susceptiblestrains were as follows. On 7H11 agar: MIC of 0.05 μg/ml was found foreight strains, and MIC of 0.2 μg/ml was found for five strains. On HSTBagar, MIC of INH was 0.2 μg/ml for all 13 strains. These data indicatedthat the highest MICs of NH for both types of agar were the same, 0.2μg/ml. The MICs of NH for ten strains previously identified asINH-resistant were as follows: eight strains on HSTB and seven strainson 7H11 agar had an MIC greater than 3.2 μg/ml, MIC of 1.6 μg/ml wasfound for one strain on both media, MIC of 0.8 μg/ml also was found forone strain on 7H11 agar only. The conclusion from these data is that theactivity of NH on HSTB was not significantly different from that on 7H11agar. These data justify the same critical concentration on both media:0.2 μg/ml for detection of the low level, and 1.0 μg/ml for detection ofthe high level of resistance to NH.

[0237] The MICs of RMP for 13 susceptible strains were as follows: on7H11 agar within the range (μg/ml) of 0.03 (1 strain)—0.125 (9strains)—0.5 (3 strains); on HSTB agar (μg/ml)—0.125 (3 strains)—0.5 (9strains)—1.0 (1 strain). These data suggest that the MICs of RMP werehigher on HSTB agar than on 7H11 agar, with one strain having 1.0 μg/ml.Results of testing 11 RMP-resistant strains were the same on both media:MIC of one strain was 4.0 μg/ml, and MICs of 10 strains were greaterthan 16.0 μg/ml. These data suggest that the critical concentration ofRMP on HSTB agar should be 2.0 μg/ml rather than 1.0 μg/ml adopted for7H11 agar. TABLE 2 MICs (mcg/ml) of INH for 13 Susceptible Strains onAgar Media HSTB 7H11 0.05 0.2 Total 0.05 0 8 8 0.2 0 5 5 Total 0 13 13MICs (mcg/ml) of INH for 10 Resistant Strains on Agar Media HSTB 7H110.4 0.8 1.6 ≧3.2 Total 0.4 0 0 1 0 1 0.8 0 1 0 0 1 1.6 0 0 0 1 1 ≧3.2 00 0 7 7 total 0 1 1 8 10

[0238] TABLE 3 MICs (mcg/ml) of RMP for 13 Susceptible Strains on AgarMedia HSTB 7H11 0.03 0.12 0.5 1.0 Total 0.03 0 1 0 0 1 0.12 0 2 7 0 90.5 0 0 2 1 3 1.0 0 0 0 0 0 TOTAL 0 3 9 1 13 MICs (mcg/ml) of RMP for 11Resistant Strains on Agar Media HSTB 7H11 4.0 ≧16.0 Total 4.0 1 0 1≧16.0 0 10 10 Total 1 10 11

Example 4

[0239] The following example describes the isolation of M. tuberculosisfrom sputum on HSTB plates.

[0240] Preparation of the agar bi-plates. Plastic bi-plates, 100×15 mm,were used for having plain (I) and selective (II) HSTB agar. Four drugsunder the PACT acronym, the same as for the 7H10/7H11 agar, were used tomake the medium selective. Stock solutions of each of these drugs wereprepared in advance. Polymyxin B (Sigma, A3800406-001) was kept in anaqueous solution of 100,000 units per ml (unit=0.127 μg). Amphotericin B(Fungizone, Sigma, A4888) was kept protected from light in an aqueoussolution containing 10,000 μg/ml. Carbenicillin (Geopen, Sigma C1389)was also kept in an aqueous solution containing 100,000 μg/ml.Trimethoprim Lactate (Sigma, T7883) was prepared in a 10,000 μg/mlaqueous solution.

[0241] The drug solutions were kept at −70° C. in 1.0-ml aliquots, andafter thawing, a mixture (PACT) was prepared in the following manner:Polymyxin B  0.5 ml Amphotericin B  0.5 ml Carbenicillin 0.25 mlTrimethoprim 0.62 ml

[0242] To make 200.0 ml of the selective agar medium, 20.0 ml of thePACT mixture combined with 20.0 ml of serum was added to 160.0 ml of thecooled agar and carefully stirred with magnetic bars to avoid bubbles.The final concentrations of drugs (μg/ml) in the selective agar mediumwere: P—200 units, A—10 μg, C—50 μg, T—20 μg. Each half of the bi-platecontained about 10.0 ml of either plain or selective agar. Aftersolidification, each plate was placed in a separate zip-lock plastic bagand kept refrigerated, protected from light.

[0243] Inoculation and Cultivation. Sputum specimens were processed bythe standard NaOH-NALC procedure (described in the Detailed Descriptionof the Invention), or by a similar procedure with pH neutralization. Thepellet obtained after concentration through centrifugation at 3500×g inan aerosol-contained refrigerated centrifuge should be diluted withbovine albumin or animal serum to have a sufficient volume to inoculateall necessary media. Bi-plates are inoculated with 0.2 ml of theconcentrated specimen per each side of the plate, and 0.11 ml is neededper each segment of the four-segment plate. Each plate is left under thehood to allow the inoculum (spread by tilting of the plate) to be fullyabsorbed by the medium. Each plate is placed into a zip-lock bag. Theplates, agar side down, are stacked no more than six plates high, inwire baskets, and incubated in regular incubators (without CO₂),protected from light. The plates are examined after three weeks ofincubation, and in case of no growth or insufficient growth, also atfour and six weeks. At any time-point, upon removal from the incubatorthe plates should be left in bags at the room temperature overnight,turned upside down (agar side up) to eliminate condensation. Thecolonies are examined and counted under dissecting microscope, withoutremoving them from the plastic bags. If more detailed examination of thecolonies is required, it should be done under the hood in the level 3biosafety facility.

[0244] Results. Twenty M. tuberculosis cultures were isolated fromsputum specimens inoculated into five culture media: BACTEC 12B broth,MGIT broth, L-J slant, 7H11 agar (cultivation with CO₂), and HSTB agar(cultivation without CO₂). The best recovery rate was on HSTB agar (19cultures), followed by BACTEC and MGIT broth (15 cultures each), 7H11agar (11 cultures), and L-J slant (9 cultures). Comparison of theresults on two agar media indicated that two isolates did not grow oneither medium, and seven cultures grew on HSTB, but not on 7H11 agar.Incorporation into the isolation protocol of the BACTEC liquid mediumwas important to supplement the results on 7H11 agar by adding six morepositive cultures totaling in 17 positive results. This outcome wasstill less than 19 cultures on HSTB agar alone (95%).

[0245] Time to growth detection was shortest in the broth systems, MGITand BACTEC: 12.1 and 14.2 mean number of days, with 50% and 30% reportedwithin two weeks. The mean periods on 7H11 and HSTB agar weresignificantly longer (17.1 and 25.1 days) due to a few strains thatrequired up to four to five weeks to produce visible growth. At the sametime, 20% and 30% of the isolates were detected on these media withinthe first two weeks of cultivation.

[0246] These results show that HSTB agar was more efficient than 7H11agar and all other media tested (L-J slant, BACTEC and MGIT broth) inrecovery from the 20 sputum specimens evaluated. Incorporation of aliquid medium into isolation protocol increases the recovery rate and toshorten the turnaround time. With HSTB agar, the addition of a liquidmedium is useful for shortening the time to detection of growth for anumber of strains. At the same time, the recovery rate on HSTB agar wasnot less than in a combined use of 7H11 agar and BACTEC broth. TABLE 4Recovery of M. tuberculosis from Sputum Specimens on Different Media No.of Positive Cultures on Smear Results No. of (AFB/field) Isolates HSTB7H11 L-J BACTEC 0 4 4 0 0 3 <5 9 8 5 4 6 ≧5 7 7 6 5 6 TOTAL 20 19 11 915

[0247] TABLE 5 Recovery of M. tuberculosis from 20 Culture-PositiveSputa: Combined Effect of 7H11 or HSTB Agar with 7H12 BACTEC Broth HSTBAgar BACTEC Pos. Neg. Total Pos. 15 0 15 Neg. 4 1 5 7H11 Agar BACTECPos. Neg. Total Pos. 9 6 15 Neg. 2 3 5 Total 11 9 20

[0248] TABLE 6 Time to Recovery of M. tuberculosis from 20Culture-Positive Sputum Specimens on Different Media Days to Detection %of Positive Medium No. of Positive Mean Range within 14 days HSTB 1925.1 7-42 30.0 7H11 11 17.1 7-28 20.0 Bactec 15 14.2 3-28 30.0 MGIT 1512.1 3-23 50.0

Example 5

[0249] The following example demonstrates that HSTB agar medium can beused with PZA to determine the actual proportion of PZA-resistantbacteria in a population. The example also demonstrates the use of HSTBagar medium in a PZA susceptibility test with clinical isolates.

[0250] The agar proportion method should provide the opportunity fordetermining the actual proportion of resistant bacteria in thepopulation. To investigate the applicability of this option to PZA, thepresent inventors conducted experiments with artificially preparedmixtures containing various proportions of PZA-resistant bacteria. Forthis purpose, PZA-resistant mutants were developed by selection from twopansusceptible strains on agar plates containing 12 μg/ml. Mixtures withthe original susceptible strains were prepared in a proportion of 10%,25% and 50%, tested along with the original strains and the resistantmutants. All five cultures were tested by two methods: in the BACTEC PZAbroth at 100, 300 and 900 μg/ml and on agar plates at 300, 900, and 1200μg/ml.

[0251] Materials and Methods

[0252] PZA was purchased from Sigma Chemical Co. (St. Louis, Mo.). Thenecessary solutions were made in distilled water. Three solutions weremade to have the final concentrations of 300, 900 and 1200 μg/ml in theagar medium (see below).

[0253] The culture medium was prepared as described in Example 2. Whenthe animal serum was added to the medium, 20 ml of the PZA-solution (ordistilled water for the control) was also added up to the total volumeof 200 ml per flask. The final concentration of the serum was 10%, andthe final pH was 6.15±0.1.

[0254] The media were poured into the 100×15 mm four-segment petridishes: No. 1—for the drug-free medium, and three remaining segments—forthe agar containing three PZA concentrations. After completion of thequality controls for sterility and ability to support growth, the plateswere stored at 4° C., protected from light, for a period not longer thaneight weeks.

[0255] A culture of M. tuberculosis was subcultivated in 7H9 broth at37° C. for a period of 4 to 7 days, and was adjusted using the samemedium to the optical density of the McFarland Standard No. 1. Twodilutions of this suspension, 10⁻² and 10⁻⁴, were used as an inoculum,0.1 ml per segment, to inoculate two plates. The plates were sealed inindividual polyethylene CO₂-permeable bags (XPEDX, Denver, Colo.), andincubated right side-up at 37° C. in the presence of 5-7% CO₂ for aperiod of 21 days. Afterwards, the plates were removed from theincubator and placed on the bench upside-down at room temperature for atleast three hours (or overnight), to eliminate the condensate. Theplates were examined without opening the polyethylene bags, using adissecting microscope (10×). The number of colonies on each segment werecounted, and the number of colonies on drug-containing segments wascompared with that on the drug-free control.

[0256] Laboratory strains (QC): M. tuberculosis H₃₇Rv susceptible to allanti-tuberculosis drugs (ATCC #27294), and M. tuberculosismono-resistant to PZA (ATCC #35828). Two PZA-resistant mutants weredeveloped from pansusceptible strains (H₃₇Rv and #9719) by selection inthe presence of 12 μg/ml of PZA on agar plates at pH 6.0. Fifty-fiveclinical isolates, reported by our clinical laboratory as susceptible(25) or resistant (30) to PZA, were tested by the BACTEC radiometricmethod in the pH 6.0 liquid medium, using three PZA concentrations—100,300, and 900 μg/ml (Heifets, In L. B. Heifets (ed.), Drug susceptibilityin the chemotherapy of mycobacterial infections, Chapter 3, pp. 89-122,CRC Press, Boca Raton, 1991). The phenotypic assessments of the resultsfor the 25 PZA-resistant strains was confirmed genetically by Dr.Zhang's laboratory (Scorpio et al., Antimicrob. Agents Chemother.,41:540-543, 1997; Scorpio et al., Nature Med., 2:662-667, 1996).

[0257] Results

[0258] The broth-determined MICs of PZA for two pansusceptible strainswere ≦100 μg/ml at pH 6.0 of the standard BACTEC PZA medium. While thegrowth of one of these strains (9719) was completely inhibited by alldrug-concentrations incorporated in the agar medium, a substantialproportion (35.9%) of another strain (H₃₇Rv) was not inhibited by 300μg/ml in agar (Table 7). Growth of both PZA-resistant mutants was notinhibited by all drug-concentrations used for both media, showing fullresistance to all concentrations used in the BACTEC broth (MIC>99μg/ml). Suspensions prepared with the intention of having 10%, 25% and50% of PZA-resistant bacteria in the mixtures of original susceptibleand their resistant mutants, have shown proportions of resistantbacteria growth on the plates approximating that in the preparedmixtures. This correlation is indicative of the potential to report theproportion of the PZA-resistant bacteria in a specimen, even if suchproportion is as low as 10%. TABLE 7 Evaluation of two PZAsusceptibility methods using artificial mixtures of resistant mutantswith the original strain Proportions (%%) of resistant bacteria as % ofresistant determined by agar plates containing the drug bacteria (μg/ml)MIC (μg/ml) incorporated 300 900 1200 Agar BACTEC a) strain H₃₇Rv 0 35.90.7 0 900 <100 10 42.7 23.8 18.9 >1200 >900 25 42.6 26.6 23.9 >1200 >90050 57.0 78.5 43.5 >1200 >900 100 98.6 74.1 96.3 >1200 >900 b) strain9719 0 0 0 0 <300 <100 10 13.8 8.6 8.6 900 >900 25 18.1 16.216.5 >1200 >900 50 35.9 36.9 40.0 >1200 >900 100 97.8 96.1 86.2 >1200>900

[0259] The results of the test in agar medium supplemented with bovinecalf serum were compared with that of the BACTEC method using differentPZA concentrations. Table 8a analyzes the results for 900 and 122 μg/mlin agar medium vs 300 or 900 μg/ml in the BACTEC medium. This analysisindicated 100% agreement for 25 PZA-susceptible strains tested witheither 900 or 1200 μg/ml incorporated in the agar medium. From a totalof 30 strains identified as resistant to 300 μg/ml by the BACTEC method,resistance to PZA in agar medium was observed for 29 strains with aconcentration of 900 μg/ml (96.7%) and for 27 strains with 1200 μg/ml(90%).

[0260] When the breakpoint of 900 μg/ml has been used in the BACTECsystem (Table 8b), the agreement in results for susceptible strains was92.9% (26 of 28 strains) or 96.4% (27 of 28 strains). Detection ofresistance by this approach was slightly better than in the previoussetting (Tables 8a and 8b). TABLE 8 Comparison of the results of testing55 M. tuberculosis clinical isolates with PZA by two methods Number ofstrains by the agar proportion method 900 μg/ml 1200 μg/ml SusceptibleResistant Susceptible Resistant a) Comparison with 300 μg/ml in BACTECBy the BACTEC 300 μg/ml Susceptible 25 0 25 0 Resistant 1 29 3 27 b)Comparison with 900 μg/ml in BACTEC By the BACTEC 900 μg/ml Susceptible26 2 27 1 Resistant 0 27 1 26

[0261] While various embodiments of the present invention have beendescribed in detail, it is apparent that modifications and adaptationsof those embodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

What is claimed is:
 1. An agar medium, comprising: a. an agar basesuitable for growth of Mycobacterium tuberculosis; and, b. animal serumat a concentration of between about 8% and 12% of the final volume ofthe agar medium; wherein said medium is at a pH of between about 6.0 toabout 6.25.
 2. The agar medium of claim 1, wherein said agar base isselected from the group consisting of Middlebrook and Cohn 7H10 andMiddlebrook and Cohn 7H11.
 3. The agar medium of claim 1, wherein saidanimal serum is selected from the group consisting of bovine fetal calfserum, calf serum, bovine serum, equine fetal calf serum and equineserum.
 4. The agar medium of claim 1, wherein said animal serum is at aconcentration of between about 9% and 11% of the final volume of theagar medium.
 5. The agar medium of claim 1, wherein said animal serum isat a concentration of about 10% of the final volume of the agar medium.6. The agar medium of claim 1, wherein said medium is at a pH of betweenabout 6.1 and 6.2.
 7. The agar medium of claim 1, wherein said medium isat a pH of about 6.1.
 8. The agar medium of claim 1, wherein said mediumfurther comprises a mixture of antimicrobial agents comprising polymixinB, carbenicillin, amphotericin B, and trimethoprim lactate.
 9. The agarmedium of claim 8, wherein said antimicrobial agents are present at afinal concentration of: 200 units/ml polymyxin B, 10 μg/ml amphotericinB, 50 μg/ml carbenicillin, and 20 μg/ml trimethoprim.
 10. The agarmedium of claim 1, wherein said medium further comprises an amount of atleast one drug effective for the identification of Mycobacteriumtuberculosis organisms that are susceptible or resistant to said drug.11. The agar medium of claim 10, wherein said at least one drug isselected from the group consisting of: isoniazid, streptomycin sulfate,di-hydro-streptomycin, rifampin, pyrazinamide, ethambutol, etionamide,capreomycin sulfate, amikacin, kanamycin sulfate, levofloxacin,p-aminosalicylic acid, D-cycloserine, and clofazimine.
 12. The agarmedium of claim 10, wherein said at least one drug comprises isoniazidand rifampin, and wherein each of said drugs is isolated within asegment of said agar medium.
 13. The agar medium of claim 12, whereinsaid isoniazid is present in two segments of said agar medium, andwherein each segment contains a different concentration of saidisoniazid.
 14. The agar medium of claim 10, wherein said at least onedrug comprises: isoniazid, rifampin, pyrazinamide and either ofstreptomycin sulfate or di-hydro-streptomycin, and wherein each of saiddrugs is isolated within a different segment of said agar medium. 15.The agar medium of claim 10, wherein said at least one drug comprises:ethambutol, etionamide, levofloxacin, capreomycin sulfate, and either ofamikacin or kanamycin sulfate, and wherein each of said drugs isisolated within a different segment of said agar medium.
 16. The agarmedium of claim 10, wherein said at least one drug comprises:p-aminosalicylic acid, D-cycloserine, and clofazimine, and wherein eachof said drugs is isolated within a different segment of said agarmedium.
 17. The agar medium of claim 10, wherein said at least one drugcomprises pyrazinamide.
 18. A method for culturing Mycobacteriumtuberculosis, comprising inoculating an agar medium with a samplecontaining Mycobacterium tuberculosis, wherein said agar mediumcomprises: a. an agar base suitable for growth of Mycobacteriumtuberculosis; and, b. animal serum at a concentration of between about8% and 12% of the final volume of the agar medium; wherein said mediumis at a pH of between about 6.0 to about 6.25.
 19. The method of claim18, wherein said medium further comprises a mixture of antimicrobialagents comprising polymixin B, carbenicillin, amphotericin B, andtrimethoprim lactate.
 20. The method of claim 19, wherein saidantimicrobial agents are incorporated into one half of said agar mediumin a plate, and wherein the other half of said agar medium in said platedoes not contain said antimicrobial agents.
 21. The method of claim 18,wherein said agar medium is inoculated with an undiluted sample obtainedfrom a patient.
 22. The method of claim 18, wherein said sample isdiluted by at least about 10 fold.
 23. The method of claim 18, whereinsaid sample is a previously isolated sample of Mycobacteriumtuberculosis.
 24. A method for testing the drug susceptibility of aculture of Mycobacterium tuberculosis, comprising: a. inoculating anagar medium with a sample containing Mycobacterium tuberculosis, whereinsaid agar medium comprises: i. an agar base suitable for growth ofMycobacterium tuberculosis; ii. animal serum at a concentration ofbetween about 8% and 12% of the final volume of the agar medium; andiii. an amount of at least one drug effective for selection againstMycobacterium tuberculosis organisms that are susceptible to said drug;wherein said medium is at a pH of between about 6.0 to about 6.25; b.incubating said inoculated agar medium for a time sufficient to detectgrowth of said Mycobacterium tuberculosis in the absence of agrowth-inhibiting drug; and, c. measuring growth of said Mycobacteriumtuberculosis on said agar medium as compared to growth of saidMycobacterium tuberculosis on said agar medium in the absence of said atleast one drug, wherein a growth rate of said Mycobacterium tuberculosison said agar medium containing said at least one drug that is less thana pre-established drug-resistance level for said at least one drug, whencompared to the growth rate of said Mycobacterium tuberculosis on saidagar medium in the absence of said at least one drug, indicates thatsaid Mycobacterium tuberculosis is susceptible to said at least onedrug.
 25. The method of claim 24, wherein said pre-establisheddrug-resistance level is 1%, and wherein a growth rate of saidMycobacterium tuberculosis on said agar medium containing said at leastone drug that is less than 1% of the growth rate of said Mycobacteriumtuberculosis on said agar medium in the absence of said at least onedrug, indicates that said Mycobacterium tuberculosis is susceptible tosaid at least one drug.
 26. The method of claim 24, wherein said atleast one drug is pyrazinamide, wherein said pre-establisheddrug-resistance level is 10%, and wherein a growth rate of saidMycobacterium tuberculosis on said agar medium containing saidpyrazinamide that is less than 110% of the growth rate of saidMycobacterium tuberculosis on said agar medium in the absence of saidpyrazinamide, indicates that said Mycobacterium tuberculosis issusceptible to pyrazinamide.
 27. The method of claim 24, wherein saidstep of incubating is for a time of at least about 3 weeks.
 28. Themethod of claim 24, wherein said step of incubating is for a time offrom between about 3 weeks and about 6 weeks.
 29. The method of claim24, wherein said step of incubating is performed in the absence ofsupplemental CO₂.
 30. The method of claim 24, wherein said at least onedrug is selected from the group consisting of: isoniazid, streptomycinsulfate, di-hydro-streptomycin, rifampin, pyrazinamide, ethambutol,etionamide, capreomycin sulfate, amikacin, kanamycin sulfate,levofloxacin, p-aminosalicylic acid, D-cycloserine, and clofazimine. 31.The method of claim 24, wherein said medium comprises the drugsisoniazid and rifampin, and wherein each of said drugs is isolatedwithin a different segment of said agar medium.
 32. The method of claim31, wherein said isoniazid is present in two different segments of saidagar medium, and wherein each segment contains a different concentrationof said isoniazid.
 33. The method of claim 31, wherein said agar mediumis directly inoculated with a sample collected from a patient.
 34. Themethod of claim 33, wherein said sample is undiluted.
 35. The method ofclaim 33, wherein said agar medium is inoculated with a sample dilutedby at least 10 fold.
 36. The method of claim 24, wherein said agarmedium comprises the drugs: isoniazid, rifampin, pyrazinamide and eitherof streptomycin sulfate or di-hydro-streptomycin, and wherein each ofsaid drugs is isolated within a different segment of said agar medium.37. The method of claim 36, wherein said agar medium is inoculated witha previously isolated culture of Mycobacterium tuberculosis from asample obtained from a patient.
 38. The method of claim 24, wherein saidmedium comprises the drugs: ethambutol, etionamide, levofloxacin,capreomycin sulfate, and either of amikacin or kanamycin sulfate, andwherein each of said drugs is isolated within a different segment ofsaid agar medium.
 39. The method of claim 38, wherein said agar mediumis inoculated with a previously isolated culture of Mycobacteriumtuberculosis from a sample obtained from a patient.
 40. The method ofclaim 24, wherein said medium comprises the drugs: p-aminosalicylicacid, D-cycloserine, and clofazimine, and wherein each of said drugs isisolated within a different segment of said agar medium.
 41. The methodof claim 40, wherein said agar medium is inoculated with a previouslyisolated culture of Mycobacterium tuberculosis from a sample obtainedfrom a patient.
 42. The method of claim 24, wherein said agar mediumcomprises the drug, pyrazinamide.